The application of molecular biological techniques, such as reverse transcription, cloning, restriction analysis, sequencing, or the like to nucleic acids, including RNAs and DNAs, requires that the nucleic acids be provided substantially free of certain contaminants. Such contaminants include, among others, substances that block or inhibit chemical reactions, including nucleic acid hybridizations, enzymatically catalyzed reactions, and other types of reactions, used in molecular biological techniques; substances that catalyze the degradation or depolymerization of a nucleic acid of interest; or substances that provide "background" indicative of the presence in a sample of a quantity of a nucleic acid of interest when the quantity is in fact not present in the sample. These contaminants include macromolecular substances, such as enzymes, other types of proteins, polysaccharides, or polynucleotides, as well as lower molecular weight substances, such as lipids, low molecular weight enzyme inhibitors or oligonucleotides.
The problem of obtaining DNA or RNA of interest sufficiently free of contaminants for application of molecular biological methods is complicated by the complex systems in which the DNA or RNA is typically found. These systems, such as cells from tissues, cells from body fluids such as blood, lymph, milk, urine, feces, semen, or the like, cells in culture, agarose or polyacrylamide gels, or solutions in which target nucleic acid amplification has been carried out, typically include significant quantities of contaminants from which the DNA or RNA of interest must be removed intact before being subjected to a molecular biological procedure.
Conventional protocols for obtaining DNA or RNA from cells are well known in the art and are described in, for example, Chapter 2 (DNA) and Chapter 4 (RNA) of F. Ausubel et al., eds., Current Protocols in Molecular Biology, Wiley-Interscience, New York (1993). For DNA, these protocols generally entail gently lysing the cells with solubilization of the DNA and enzymatically or chemically substantially freeing the DNA from contaminating substances such as proteins, RNA and other substances (i.e., reducing the concentrations of these contaminants in the same solution as the DNA to a level that is low enough that the molecular biological procedures of interest can be carried out). For isolation of RNA, the lysis and solubilization procedures must include measures for inhibition of ribonucleases and contaminants to be separated from the RNA including DNA.
The protocols also generally entail use of phenol/chloroform extraction (i.e., extraction with phenol/chloroform or phenol/chloroform/isoamyl alcohol) and ethanol (or isopropanol) precipitation (to obtain DNA) that is substantially free of contaminants that would interfere with molecular biological procedures with the DNA. However, phenol/chloroform extractions have significant drawbacks. Among these drawbacks are the time required for the multiple steps necessary in the extractions and the dangers of using phenol or chloroform. Phenol causes severe burns on contact. Chloroform is highly volatile, toxic and flammable. These dangers require that phenol be handled and phenol/chloroform extractions be carried out in a fume hood. Another undesirable characteristic of phenol/chloroform extractions is that the oxidation products of phenol can damage nucleic acids. Only freshly redistilled phenol can be used effectively, and nucleic acids cannot be left in the presence of phenol. Generally also, multi-step procedures are required to isolate RNA after phenol/chloroform extraction. Ethanol (or isopropanol) precipitation must be employed to precipitate the DNA from a phenol/chloroform-extracted aqueous solution of DNA and remove residual phenol and chloroform from the DNA. Further, ethanol (or isopropanol) precipitation is required to remove some nucleoside triphosphate and short (less than about 30 bases or base pairs) single or double-stranded oligonucleotide contaminants from the DNA.
There is a need recognized in the art for methods, that are simpler, safer, or more effective than phenol/chloroform extraction/ethanol precipitation to purify DNA sufficiently for manipulation using molecular biological procedures.
There further is a need in the art for improved methods of isolation of RNA to a purity sufficient for manipulation.
Fractionation according to size of DNA recovered from cells is required for many molecular biological procedures, and such fractionation is typically accomplished by agarose or polyacrylamide gel electrophoresis. For analysis or treatment by a molecular biological procedure after fractionation, the DNA in the fraction(s) of interest must be separated from contaminants, such as agarose, other polysaccharides, polyacrylamide, acrylamide, or acrylic acid, in the gel used in such electrophoresis. Thus, there is a need in the art for methods to accomplish such separations.
Methods for amplifying nucleic acids or segments thereof, such as the well known polymerase chain reaction (PCR) process (see, e.g., U.S. Pat. No. 4,683,202), yield solutions of complex mixtures of enzymes, nucleoside triphosphates, oligonucleotides, and other nucleic acids. Typically, the methods are carried out to obtain an highly increased quantity of a single nucleic acid segment ("target segment"). Often it is necessary to separate this nucleic acid from other components in the solution after the amplification process has been carried out. There is a need in the art for simple methods to accomplish these separations.
A particular problem in this regard that sometimes arises in amplifications by the PCR method is the problem of separating the nucleic acid (usually double-stranded DNA) intended to be amplified in the process from "primer dimers," that also might be amplified to a high level in the process. "Primer dimers" are DNAs which result from priming of DNA synthesis by one of the primers used in a PCR amplification on another primer as a template. Primer dimers can amplify to high concentrations in PCR amplifications.
Silica materials, including glass particles, such as glass powder, silica particles, and glass microfibers prepared by grinding glass fiber filter papers, and including diatomaceous earth have been employed in combination with aqueous solutions of chaotropic salts to separate DNA from other substances and render the DNA suitable for use in molecular biological procedures by substantially freeing the DNA of contaminants that would impair or prevent its being employed in such procedures. See U.S. Pat. No. 5,075,430 and references cited therein, including Marko et al., Anal. Biochem. 121, 382-387 (1982) and Vogelstein et al., Proc. Natl. Acad. Sci. (USA) 76, 615-619 (1979). See also Boom et al., J. Clin. Microbiol. 28, 495-503 (1990). With reference to intact glass fiber filters used in combination with aqueous solutions of a chaotropic agent to separate DNA from other substances, see Chen and Thomas, Anal. Biochem. 101, 339-341 (1980). Vogelstein et al., supra, suggest that silica gel is not suitable for use in DNA separations. With regard to separation of RNA using silica materials and chaotropic agents, see Gillespie et al., U.S. Pat. No. 5,155,018.
Prior to the present invention, mixtures of glass particles and silica gel, such mixtures in suspension in aqueous solutions of chaotropic agents, and the use of such suspensions to separate DNA or RNA from other substances or isolate DNA or RNA so separated were not known.